U.S. patent application number 13/611419 was filed with the patent office on 2013-01-03 for synergistic therapeutic use of prothrombin complex concentrates with fvlll concentrates.
This patent application is currently assigned to CSL Behring GmbH. Invention is credited to Gerhard DICKNEITE.
Application Number | 20130005657 13/611419 |
Document ID | / |
Family ID | 38137453 |
Filed Date | 2013-01-03 |
United States Patent
Application |
20130005657 |
Kind Code |
A1 |
DICKNEITE; Gerhard |
January 3, 2013 |
SYNERGISTIC THERAPEUTIC USE OF PROTHROMBIN COMPLEX CONCENTRATES
WITH FVlll CONCENTRATES
Abstract
The field of the invention is the treatment of acquired
bleeding, a clinical condition associated with severe traumatic,
peri- or post-operative bleeding. A novel treatment is proposed in
which synergistic pro-coagulatory properties of Prothrombin Complex
Concentrates (PCC) together with medicaments comprising FVIII
and/or vWF are exploited.
Inventors: |
DICKNEITE; Gerhard;
(Marburg, DE) |
Assignee: |
CSL Behring GmbH
|
Family ID: |
38137453 |
Appl. No.: |
13/611419 |
Filed: |
September 12, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12520773 |
Jun 22, 2009 |
|
|
|
PCT/EP2007/011099 |
Dec 18, 2007 |
|
|
|
13611419 |
|
|
|
|
Current U.S.
Class: |
514/14.1 ;
514/13.7 |
Current CPC
Class: |
A61K 38/36 20130101;
A61K 38/37 20130101; A61K 38/4846 20130101; A61P 43/00 20180101;
A61P 7/00 20180101; A61P 7/04 20180101; A61P 7/02 20180101 |
Class at
Publication: |
514/14.1 ;
514/13.7 |
International
Class: |
A61K 38/36 20060101
A61K038/36; A61P 7/04 20060101 A61P007/04; A61K 38/37 20060101
A61K038/37 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 2006 |
EP |
06026748.1 |
Claims
1.-5. (canceled)
6. A kit for treatment of acquired coagulopathy in a patient
comprising a first pharmaceutical composition comprising isolated
coagulation factors FII, FIX, FX and FVII and a second
pharmaceutical composition comprising isolated FVIII and/or
isolated vWF.
7. The kit according to claim 6, wherein the isolated coagulation
factors FII, FIX, FX and FVII of the first composition are a
prothrombin complex concentrate (PCC) derived from human blood.
8. The kit according to claim 6, wherein the isolated coagulation
factors FII, FIX, FX and FVII of the first composition are
recombinantly expressed, and wherein the recombinantly expressed
coagulation factors are combined such that the ratios of antigen
and activity of said recombinantly expressed coagulation factors
correspond to PCCs derived from human blood.
9. The kit according to claim 6, wherein the isolated coagulation
factor(s) FVIII and/or vWF of the second composition are derived
from human blood.
10. The kit according to claim 6, wherein the isolated coagulation
factor(s) FVIII and/or vWF of the second composition are
recombinantly expressed.
11. The kit according to claim 6, wherein the first composition
comprises 5-180 lU/ml FII, 2.5-100 lU/ml FVII, 5-120 lU/ml FIX, and
5-240 lU/ml FX, and the second composition comprises 5-120 lU/ml
FVIII and/or 13-320 lU/ml vWF.
12. A method of treating acquired coagulopathy comprising
administering to a patient in need thereof an effective amount of
the kit according to claim 6.
13. The method according to claim 12, wherein the acquired
coagulopathy is one or more of bleeding due to trauma; bleeding
during surgical procedures; bleeding due to cardiac surgery;
intracerebral hemorrhage; subarachnoid hemorrhage; sub- or epidural
bleeding; bleeding due to blood loss and hemodilution; bleeding due
to disseminated intravascular coagulation (DIC) and a consumption
coagulopathy; thrombocytopenia; thrombocyte dysfunction; bleeding
due to liver cirrhosis, liver dysfunction and/or fulminant liver
failure; bleeding due to liver biopsy in a patient with liver
disease; bleeding after organ transplantation; bleeding from
gastric varices; peptic ulcer bleeding; gynecological bleeding;
periventricular hemorrhage in a low birth weight child; post partum
haemorrhage; bleeding due to fatal distress of a newborn; bleeding
associated with a burn; bleeding associated with amyloidosis;
bleeding due to hematopoietic stem cell transplantation associated
with a platelet disorder; bleeding associated with malignancy;
bleeding due to infection with a hemorrhagic virus; and bleeding
associated with pancreatitis.
14. The method according to claim 13, wherein the acquired
coagulopathy is bleeding due to trauma.
15. The method according to claim 12, wherein the first and second
compositions of the kit are administered simultaneously.
16. The method according to claim 12, wherein the first and second
compositions of the kit are administered separately.
17. The method according to claim 12, wherein the first and second
compositions of the kit are administered sequentially.
18. The method according to claim 12, wherein the first composition
of the kit comprises 5-180 lU/ml FII, 2.5-100 lU/ml FVII, 5-120
lU/ml FIX, and 5-240 lU/ml FX, and the second composition of the
kit comprises 5-120 lU/ml FVIII and/or 13-320 lU/ml vWF.
Description
[0001] The field of the invention is the treatment of acquired
bleeding, a clinical condition associated with severe traumatic,
peri- or postoperative bleeding. A novel treatment is proposed in
which synergistic procoagulatory properties of Prothrombin Complex
Concentrates (PCC) together with medicaments comprising FVIII
and/or vWF are exploited.
[0002] Bleeding as a result of an acquired coagulopathy without an
underlying genetic disorder can occur in a variety of clinical
constellations. Trauma, peri- or postoperative hemorrhage or
anticoagulant overdose can impair the coagulation system. In severe
cases coagulopathy is associated with massive blood loss, which
needs to be corrected by volume replacement and/or erythrocyte
transfusion. Dilution of the functional elements of the coagulation
system leads to an elevated bleeding risk. Besides surgical wound
closing, restitution of the patient's impaired hemostatic potential
is mandatory (Hardy, 2006). Hemostasis is a complex system to
prevent the loss of circulating blood. Its main elements are the
plasmatic coagulation factors and the circulating platelet pool.
The initial event after an injury is the binding of the platelet to
the newly exposed subendothelial collagen via its GPIb receptor.
This binding is mediated by von Willebrand factor (vWF), a large
multimeric protein. VWF has a binding site (A1) for the platelet
GPIb receptor, the A3 domain is responsible for the binding to
collagen (Ruggeri, 1993). The exposed tissue factor in the
subendothelium binds circulating FVII and activates the extrinsic
clotting systems by forming activated FVIIa in a presently unknown
fashion (Morrissey, 2001). The TF/FVIIa complex activates Factor X
to FXa to form a small amount of thrombin. This initial thrombin
activates platelets at the site of injury and also the coenzymes FV
and FVIII to FVa and FVIIIa, respectively. FXa then bihds to the
activated platelet where it is converted to FXa by the FIXa/FVIIIa
complex. The platelet bound FXa/Va complex converts prothrombin
into thrombin and thus mediates the thrombin burst (Monroe et al.,
2002). A dilutional coagulopathy is caused by consumption, dilution
and losses and affects all aspects of coagulation: The enzymes and
proenzymes of the coagulation cascade, fibrinogen and the
thrombocytes (Hiippala et al., 1995, Hiippala 1998). As
erythrocytes also contribute to hemostasis a decrease of the
hematocrite also impairs hemostasis (McLoughlin et al., 1996,
Sheiner, 2005). The use of colloids is also known to lead to
decreased fibrin polymerization (Innerhofer et al., 2002).
Hypothermia has been shown to impair the coagulation as shown in
human plasma at different temperatures (Rohrer, 1992). Martini et
al. (2005) demonstrated in a preclinical study that hypothermia
caused a delay in the thrombin generation. Together with acidosis,
hypothermia and coagulopathy form the "lethal trial" of trauma
(Mikhail, 1999), a life-threatening coagulopathy develops in the
affected patients (Cosgriff et. al, 1997). Activation of the
fibrinolytic system might aggravate the situation by the
consumption of fibrinogen and fibrin (Vorweg et al., 2001).
[0003] The substitution with coagulation factors and cellular
elements as erythrocytes and platelets is mandatory to restore the
hemostatic balance (Spivey, 2005, Spahn, 2004). The widespread use
of fresh frozen plasma (FFP) to correct a coagulopathy is in
contrast to the little evidence for effectiveness in the published
literature (for review see Stanworth, 2004). The use of FFP is
sometimes associated with the development of transfusions related
to acute lung injury (TRALI, Bux, 2005). Cryoprecipitate from
frozen plasma is used widely in this setting. Based on the poor
efficacy and safety concerns, the search for new concepts to
overcome a dilutional coagulopathy, therefore remains a clinical
challenge.
[0004] The invention is based on the use of PCC in this clinical
setting. PCC in combination with various coagulation factors has
been proposed earlier as an antidote against different
anticoagulants e.g. against hirudin (EP 0700684). Also
pharmaceutical compositions comprising at least 2 highly purified
components selected from the group consisting of factors II, V, Va,
X and Xa optionally in combination with other coagulation factors
have been disclosed for the treatment of hemophilia A inhibitor
patients (EP0796623).
[0005] In contrast to the above mentioned PCCs which comprise
non-activated coagulation factors FII, FVII, FIX and FX, in the
clinical setting also activated PCCs are used. Examples of those
activated PCCs are FEIBA.RTM. (Immuno-Baxter) and Autoplex.RTM.
(Hyland). These activated PCCs comprise the coagulation factors
FII, FVII, FIX and FX in activated form. The use of activated PCCs
carries the inherent risk of prothrombotic side effects, which
makes the use of non-activated PCCs favourable.
[0006] In a model of dilutional coagulopathy resulting from
intended blood loss and fluid replacement recently a combination of
PCC and fibrinogen was found to be beneficial (Fries et al.,
2006).
[0007] However in view of considerable morbidity and mortality
caused by acquired coagulopathy there is a high clinical interest
and strong incentive to further increase the efficacy of treatment
of such clinical conditions. The object of the present invention is
thus to provide further improved therapies for the treatment of
acquired coagulopathies.
[0008] It was surprisingly found that a treatment of acquired
coagulopathy with isolated PCC in combination with a pharmaceutical
preparation comprising isolated FVIII and/or isolated vWF leads to
a synergistically largely increased efficacy as compared to
treatment with either PCC or a preparation comprising FVIII and
vWF. The preparation to be combined with isolated PCC comprises
FVIII and/or vWF and optionally other components which my also
contribute to hemostasis. One embodiment of the invention is the
combination of an isolated PCC with a medicament consisting
essentially of FVIII and vWF.
[0009] "Isolated" in the sense of this inventions means, that the
respective coagulation factor or mixture of coagulation factors has
been purified from either human plasma or, if produced
recombinantly, from the culture medium. Purified in the sense of
this invention means any type of purification which leads to a
higher biological activity of said coagulation factor or mixture of
coagulation factors per mg of total protein content or to a higher
biological activity of said coagulation factor or mixture of
coagulation factors per ml of liquid which is finally administered
to the patient, as compared to the solution from which the
respective coagulation factor or mixture of coagulation factors was
obtained originally.
[0010] "Simultaneous use" in the sense of the invention means that
the composition comprising isolated coagulation factors FII, FIX,
FX and FVII and the composition comprising isolated FVIII and/or
isolated vWF are mixed and then administered as a mixture to a
patient.
[0011] "Separate use" in the sense of the invention means that the
composition comprising isolated coagulation factors FII, FIX, FX
and FVII and the composition comprising isolated FVIII and/or
isolated vWF are administered both at the same time or separately
one after the other, whereby the sequence of said administrations
is not relevant.
[0012] "Sequential use" in the sense of the invention means than
the composition comprising isolated coagulation factors FII, FIX,
FX and FVII and the composition comprising isolated FVIII and/or
isolated vWF are administered separately, whereby the sequence of
said administration is not relevant, and whereby the time interval
between both administrations is at most 2 days, preferentially at
most 1 day and more preferentially at most 4 hours.
[0013] An animal model of a dilutional coagulopathy in hypothermic,
normotensive pigs by fractionated blood withdrawal and substitution
with hydroxyethyl starch (HES) was used to investigate efficacy of
the proposed treatment. A trauma was induced by an incision into
the spleen, blood loss and time to hemostasis were determined.
[0014] Substitution therapy was performed with Beriplex P/N.RTM.
and either Helixate.RTM. or Humate P.RTM.. Beriplex P/N.RTM. is a
prothrombin complex concentrate (PCC) comprising the vitamin K
dependent coagulation factors II, VII, IX and X (Schulman, 2007).
In addition Beriplex P/N.RTM. contains protein C and protein S.
Helixate.RTM. consists of recombinant FVIII as the active
constituent. Humate P.RTM., is a product consisting essentially of
FVIII as well as of vWF.
[0015] A dilutional coagulopathy was induced by the stepwise
withdrawal of about 60% of the circulating blood volume and
erythrocyte retransfusion. The decrease in circulating coagulation
factors was monitored. The function of the clotting system was
detected by thromboelastography and prothrombin time (PT). Platelet
function was monitored by aggregation and adhesion. A substitution
study consisted of the following groups: Untreated control group
(no dilutional coagulopathy), dilutional coagulopathy without
treatment, PCC, PCC+fibrinogen, PCC+vWF/FVIII, PCC+rec. FVIII,
vWF/FVIII, rec. FVIIa, FIX/FX and PCC+vWF. Restoration of the
coagulation system and bleeding after a spleen injury was
determined.
[0016] The coagulopathy led to a decrease in coagulation factors to
about 30% of the baseline concentration. Platelet numbers were
decreased from about 400.000 to 100.000/.mu.l, in addition,
aggregation and adhesion were impaired. PCC could substitute the
lacking prothrombin factors (II, VII, IX and X) and improved
coagulation. After spleen injury PCC significantly reduced time to
hemostasis and reduced blood loss when compared to the dilutional
control. On the other hand vWF/FVIII alone did not change the
outcome of the study, nor did the treatment with FIX or
respectively FVIIa. In contrast the combination of PCC with either
FVIII or vWF or vWF/FVIII significantly reduced time to hemostasis
and blood loss to nearly normal values. This shows that the
combination of FVIII/ and/or vWF with PCC is significantly more
effective than the monotherapy with PCC alone, indicating a
synergistic effect.
[0017] In the group treated with vWF/FVIII in addition the adhesion
capacity of platelets was significantly increased, indicating that
also the impaired platelet function could be normalized.
[0018] In recent years attempts have been made to substitute in
bleeding conditions with a single component of the prothrombin
complex namely the activated form of FVII which is available as
rec. FVIla (for a review see Holcomb, 2005). Rec. FVIIa is able to
directly bind to the activated platelet and to initiate the
conversion of FX to FXa while bypassing the tenase (FIXa/Villa)
pathway. This is the basis for the well-known efficacy of rec.
FVIla in treating hemophilia A and B patients with inhibitors to
FVIII or FIX (Habermann et al., 2004). While hemophilia inhibitor
patients have enough FX and prothrombin, the situation in a patient
with a severe dilution coagulopathy is different. If FX decreases
under a critical level rec. FVIla will lack its substrate. A
multicomponent therapeutic agent such as PCC can offer advantages
in a situation of a general decrease in coagulation factors. PCC
substituted for the proteins of the prothrombin complex, factors
II, VII, IX and X. The first and foremost task of the coagulation
system is to assure thrombin generation. Thrombin has multiple
functions in the coagulation system: the formation of fibrin from
fibrinogen, the activation of the cofactors V and VIII, the
activation of FXIII and the activation of platelet via the thrombin
receptor. The generation of thrombin requests sufficient
prothrombin (FII) as a substrate from the prothrombinase complex
which contains the activated form of FX as the acting serine
protease. The activated form of FIX is the enzyme of the tenase
complex, which together with FVIII converts FX to FXa. It is
tempting to believe that all four enzymes of the prothrombin
complex are indispensable to generate thrombin. Our data suggest
that it is mandatory to supply enough prothrombin complex factors
to initiate the thrombin burst and that the combination of PCC with
FVIII and/or vWF comprising compositions effectively reconstitutes
the impaired coagulation cascade and significantly shortens time to
hemostasis.
[0019] The invention relates to two pharmaceutical compositions
which, when combined show a synergistic effect.
[0020] The first pharmaceutical composition comprising isolated
coagulation factors FII, FIX, FX and FVII to be used in the present
invention and the second pharmaceutical composition comprising
isolated Will and/or isolated vWF, can be manufactured as two
independent compositions, which can be separately sold, or can be
combined in one kit as separate pharmaceutical compositions or can
be manufactured to be comprised in one single pharmaceutical
composition as a mixture of all components.
[0021] The invention therefore relates to a composition comprising
isolated coagulation factors FII, FIX, FX and FVII and a
composition comprising isolated FVIII and/or isolated vWF for
simultaneous, separate or sequential use in a method of treatment
of acquired bleeding.
[0022] The invention further relates to the use of a composition
comprising isolated coagulation factors FII, FIX, FX and FVII and a
composition comprising isolated FVIII and/or vWF for the
manufacture of pharmaceutical preparations for simultaneous,
separate or sequential use in the therapy of acquired
coagulopathies.
[0023] A further aspect of the invention is the use of a
composition comprising isolated coagulation factors FII, FIX, FX
and FVII and a composition comprising isolated FVIII and/or vWF for
the manufacture of a combined pharmaceutical preparation for
simultaneous, separate or sequential use in the therapy of acquired
coagulopathies. All compositions of the invention comprise
coagulation factors which have not been activated.
[0024] In a preferred embodiment of the invention the isolated
coagulation factors FII, FIX, FX and FVII are a prothrombin complex
concentrate (PCC) derived from human blood or a PCC reconstituted
from recombinantly expressed coagulation factors wherein the ratios
of antigen and activity of said recombinantly expressed coagulation
factors FVII, FIX, FX and FVII correspond to PCCs derived from
blood.
[0025] Prothrombin complex concentrate (PCC) in the meaning of the
present invention comprises a combination of coagulation factors
FII, FIX, FX and FVII. PCC may also contain protein C and protein
S.
[0026] Isolated PCC compositions as listed in Table I are
encompassed by the present invention. Fresh frozen plasma contains
per definition 1 lU/ml, thus the concentration of the prothrombin
complex factors are enriched several fold in PCC.
[0027] Isolated PCC in the sense of the invention encompasses PCC
compositions in which each individual coagulation factor is present
in a liquid or if stored lyophilized in the liquid after
reconstitution prior to injection by at least a factor of 2.5 as
compared to its concentration in blood.
TABLE-US-00001 TABLE 1 Compositions of preferred PCCs Most
preferred More preferred Preferred a) essential components Factor
II 20-48 IU/ml 10-90 IU/ml 5-180 IU/ml Factor VII 10-25 IU/ml 5-50
IU/ml 2.5-100 IU/ml.sup. Factor IX 20-35 IU/ml 10-60 IU/ml 5-120
IU/ml Factor X 22-60 IU/ml 10-120 IU/ml 5-240 IU/ml b) optional
components Protein C 15-45 IU/ml 8-90 IU/ml 4-180 IU/ml Protein S
13-26 IU/ml 6-50 IU/ml 3-100 IU/ml
[0028] Functional coagulation factor II (FII) displays the
biological activity of prothrombin, which represents the inactive
proenzyme of thrombin (FIIa). After activation of the coagulation
cascade the conversion of prothrombin to thrombin takes place, the
latter multiple activating functions in the coagulation system
include among others the conversion of fibrinogen to fibrin,
activation of coagulation factor XIII (FVIII) to activated
coagulation factor XIII (XIIIa), activation of FV and FVIII to FVa
and Villa, platelet activation after partial proteolysis of the
thrombin receptor.
[0029] Functional coagulation factor IX (FIX) displays the
biological activity of inactive FIX, which is converted upon
coagulation activation to the active FIXa. FIXa forms a complex
with its coenzyme FVIIIa and represents the tenase complex, which
cleaves the inactive FX to its active form FXa.
[0030] Functional coagulation factor X (FX) displays the biological
activity of inactive FX which is converted to active FXa after
coagulation activation. FXa forms a complex with its coenzyme FVa
which represents the prothrombinase complex which cleaves the
inactive prothrombin (FII) into the active thrombin (FIIa).
[0031] Functional coagulation factor FVII (FVII) displays the
biological activity of inactive FVII which is converted during the
activation of coagulation to FVIIa. FVIIa together with tissue
factor converts the inactive FX to the active FXa. Additionally
FVIla can convert inactive FIX to active FIXa.
[0032] Functional coagulation factor VIII (FVIII) displays the
biological activity of the coenzyme FVIII, which is converted to
FVIIIa during coagulation activation. FVIIIa is the coenzyme for
the protease FIXa and forms a complex with FIXa. The FVIIIa/FIXa
complex cleaves the inactive FX to form activated FXa.
[0033] Isolated FVIII in the sense of the invention encompasses
FVIII compositions in which FVIII is present in a liquid or if
stored lyophilized in the liquid after reconstitution prior to
injection in a concentration which is by at least a factor of 2.5
higher than the concentration of FVIII in blood.
[0034] Functional von Willebrand factor (vWF) displays the
biological activity of vWF a large multimeric protein with multiple
binding sites responsible for positioning thrombocytes to the site
of injury. VWF binds to the thrombocyte via its Al domain and to
the collagen at the site of injury with its A3 domain. It thus
mediates thrombocyte adhesion. Additionally thrombocyte aggregation
is induced by binding of vWF to the .alpha.IIb/.beta.3 receptor and
vWF stabilizes the circulating FVIII.
[0035] Isolated vWF in the sense of the invention encompasses vWF
compositions in which vWF is present in a liquid or if stored
lyophilized in the liquid after reconstitution prior to injection
in a concentration which is by at least a factor of 2.5 higher than
the concentration of vWF in blood.
[0036] The activity of the coagulation factors discussed above can
be measured according to L. Thomas: Clinical Laboratory
Diagnostics, TH-Books, Frankfurt, 1998, Chapter 17.
[0037] Preferred compositions of a combination preparation
comprising FVIII and vWF are indicated in table 2:
TABLE-US-00002 TABLE 2 Compositions of preferred combination
preparations comprising FVIII and vWF Most preferred More Preferred
Preferred Factor VIII 20-40 IU/ml 10-60 IU/ml 5-120 VWF 50-100
IU/ml 25-160 IU/ml 13-320
TABLE-US-00003 TABLE 3 Preferred combinations between PCC and
FVIII/vWF (X.sub.n represents respectively one out of nine
preferred combinations of a preferred PCC concentration and a
preferred FVIII/vWF concentration) Preferred FVIII/vWF
concentration Preferred PCC concentration (based on FIX units)
(based on FVIII Most preferred More preferred Preferred units)
20-35 IU/ml 10-60 IU/ml 5-120 IU/ml Most preferred X.sub.1 X.sub.2
X.sub.3 20-40 IU/ml More preferred X.sub.4 X.sub.5 X.sub.6 10-60
IU/ml Preferred X.sub.7 X.sub.8 X.sub.9 5-120 IU/ml
[0038] The coagulation factors used in said pharmaceutical
compositions can be obtained from human plasma or serum or
recombinantly. "Coagulation factors" as used in the present
invention comprise proteins that have the amino acid sequence of
native human coagulation factors. Also comprised are coagulation
factors with a slightly modified amino acid sequence, for instance,
a modified N-terminal end including N-terminal amino acid deletions
or additions so long as those proteins substantially retain the
activity of the coagulation factors. "The coagulation factors"
within the above definition also comprise natural allelic
variations that may exist and occur from one individual to another.
"The coagulation factors" within the above definition further
comprise variants of such coagulation factors. Such variants differ
in one or more amino acid residues from the wild type sequence.
Examples of such differences may include truncation of the N-
and/or C-terminus by one or more amino acid residues (e.g. 1 to 10
amino acid residues), or addition of one or more extra residues at
the N- and/or C-terminus, e.g. addition of a methionine residue at
the N-terminus, as well as conservative amino acid substitutions,
i.e. substitutions performed within groups of amino acids with
similar characteristics, e.g. (1) small amino acids, (2) acidic
amino acids, (3) polar amino acids, (4) basic amino acids, (5)
hydrophobic amino acids, (6) aromatic amino acids. Examples of such
conservative substitutions are shown in the following table.
TABLE-US-00004 (1) Alanine Glycine (2) Aspartic acid Glutamic acid
(3a) Asparagine Glutamine (3b) Serine Threonine (4) Arginine
Histidine Lysine (5) Isoleucine Leucine Methionine Valine (6)
Phenylalanine Tyrosine Tryptophane
[0039] "Functional coagulation factors" as used in this invention
comprise coagulation factor molecules displaying biological
activity either in solution and/or on cellular surfaces as
described above.
[0040] The term "recombinant" means, for example, that the variant
has been produced in a host organism by genetic engineering
techniques.
[0041] The host cells of the invention may be employed in a method
of producing human coagulation factors. The method comprises:
[0042] a) culturing host cells of the invention under conditions
such that one or more human coagulation factors is/are expressed;
and [0043] b) optionally recovering one or more human coagulation
factors from the host cells or from the culture medium.
[0044] Degree and location of glycosylation or other
post-translation modifications may vary depending on the chosen
host cells and the nature of the host cellular environment. When
referring to specific amino acid sequences, posttranslational
modifications of such sequences are encompassed in this
application.
[0045] The production of recombinant proteins at high levels in
suitable host cells, requires the assembly of the above-mentioned
modified cDNAs into efficient transcriptional units together with
suitable regulatory elements in a recombinant expression vector,
that can be propagated in various expression systems according to
methods known to those skilled in the art. Efficient
transcriptional regulatory elements could be derived from viruses
having animal cells as their natural hosts or from the chromosomal
DNA of animal cells. Preferably, promoter-enhancer combinations
derived from the Simian Virus 40, adenovirus, BK polyoma virus,
human cytomegalovirus, or the long terminal repeat of Rous sarcoma
virus, or promoter-enhancer combinations including strongly
constitutively transcribed genes in animal cells like beta-actin or
GRP78 can be used. In order to achieve stable high levels of mRNA
transcribed from the cDNAs, the transcriptional unit should contain
in its 3'-proximal part a DNA region encoding a transcriptional
termination-polyadenylation sequence. Preferably, this sequence is
derived from the Simian Virus 40 early transcriptional region, the
rabbit beta-globin gene, or the human tissue plasminogen activator
gene.
[0046] The cDNAs are then integrated into the genome of a suitable
host cell line for expression of the coagulation factors.
Preferably this cell line should be an animal cell-line of
vertebrate origin in order to ensure correct folding, Gla-domain
synthesis, disulfide bond formation, asparagine-linked
glycosylation, 0-linked glycosylation, and other post-translational
modifications as well as secretion into the cultivation medium.
Examples of other post-translational modifications are
hydroxylation and proteolytic processing of the nascent polypeptide
chain. Examples of cell lines that can be used are monkey
COS-cells, mouse L-cells, mouse C127-cells, hamster BHK-21 cells,
human embryonic kidney 293 cells, and preferentially hamster
CHO-cells. Due to its complex post-translational modifications
recombinant The coagulation factors is preferably expressed in
human cell lines.
[0047] The recombinant expression vector encoding the corresponding
cDNAs can be introduced into an animal cell line in several
different ways. For instance, recombinant expression vectors can be
created from vectors based on different animal viruses. Examples of
these are vectors based on baculovirus, vaccinia virus, adenovirus,
and preferably bovine papilloma virus.
[0048] The transcription units encoding the corresponding DNAs can
also be introduced into animal cells together with another
recombinant gene, which may function as a dominant selectable
marker in these cells in order to facilitate the isolation of
specific cell clones, which have integrated the recombinant DNA
into their genome. Examples of this type of dominant selectable
marker genes are Tn5 amino glycoside phosphotransferase, conferring
resistance to geneticin (G418), hygromycin phosphotransferase,
conferring resistance to hygromycin, and puromycin acetyl
transferase, conferring resistance to puromycin. The recombinant
expression vector encoding such a selectable marker can reside
either on the same vector as the one encoding the cDNA of the
desired protein, or it can be encoded on a separate vector which is
simultaneously introduced and integrated into the genome of the
host cell, frequently resulting in a tight physical linkage between
the different transcription units.
[0049] Other types of selectable marker genes, which can be used
together with the cDNA of the desired protein, are based on various
transcription units encoding dihydrofolate reductase (dhfr). After
introduction of this type of gene into cells lacking endogenous
dhfr-activity, preferentially CHO-cells (DUKX-B11, DG-44) it will
enable these to grow in media lacking nucleosides. An example of
such a medium is Ham's F12 without hypoxanthine, thymidin, and
glycine. These dhfr-genes can be introduced together with the
coagulation factor cDNA transcriptional units into CHO-cells of the
above type, either linked on the same vector or on different
vectors, thus creating dhfr-positive cell lines producing
recombinant protein.
[0050] If the above cell lines are grown in the presence of the
cytotoxic dhfr-inhibitor methotrexate, new cell lines resistant to
methotrexate will emerge. These cell lines may produce recombinant
protein at an increased rate due to the amplified number of linked
dhfr and the desired protein's transcriptional units. When
propagating these cell lines in increasing concentrations of
methotrexate (1-10000 nM), new cell lines can be obtained which
produce the desired protein at very high rate.
[0051] The above cell lines producing the desired protein can be
grown on a large scale, either in suspension culture or on various
solid supports. Examples of these supports are micro carriers based
on dextran or collagen matrices, or solid supports in the form of
hollow fibres or various ceramic materials. When grown in cell
suspension culture or on micro carriers the culture of the above
cell lines can be performed either as a bath culture or as a
perfusion culture with continuous production of conditioned medium
over extended periods of time. Thus, according to the present
invention, the above cell lines are well suited for the development
of an industrial process for the production of the desired
recombinant proteins
[0052] The recombinant protein, which accumulates in the medium of
secreting cells of the above types, can be concentrated and
purified by a variety of biochemical and chromatographic methods,
including methods utilizing differences in size, charge,
hydrophobicity, solubility, specific affinity, etc. between the
desired protein and other substances in the cell cultivation
medium.
[0053] An example of such purification is the adsorption of the
recombinant protein to a monoclonal antibody, which is immobilised
on a solid support. After desorption, the protein can be further
purified by a variety of chromatographic techniques based on the
above properties.
[0054] It is preferred to purify the coagulation factors of the
present invention, irrelevant whether produced by recombinant means
or obtained from human plasma, to 60% purity, more preferably
.gtoreq.80% purity, and particularly preferred is a
pharmaceutically pure state that is greater than 95% pure with
respect to contaminating macromolecules, particularly other
proteins and nucleic acids, and free of infectious and pyrogenic
agents.
[0055] The coagulation factors as described in this invention can
be formulated into pharmaceutical preparations for therapeutic use.
The purified protein may be dissolved in conventional
physiologically compatible aqueous buffer solutions to which there
may be added, optionally, pharmaceutical excipients to provide
pharmaceutical preparations.
[0056] Such pharmaceutical carriers and excipients as well as
suitable pharmaceutical formulations are well known in the art (see
for example "Pharmaceutical Formulation Development of Peptides and
Proteins", Frokjaer et al., Taylor & Francis (2000) or
"Handbook of Pharmaceutical Excipients", 3.sup.rd edition, Kibbe et
al., Pharmaceutical Press (2000)). In particular, the
pharmaceutical composition comprising the polypeptide variant of
the invention may be formulated in lyophilized or stable soluble
form. The polypeptide variant may be lyophilized by a variety of
procedures known in the art. Lyophilized formulations are
reconstituted prior to use by the addition of one or more
pharmaceutically acceptable diluents such as sterile water for
injection or sterile physiological saline solution.
[0057] Formulations of the composition are delivered to the
individual by any pharmaceutically suitable means of
administration. Various delivery systems are known and can be used
to administer the composition by any convenient route.
Preferentially the compositions of the invention are administered
systemically. For systemic use, the coagulation factors of the
invention are formulated for parenteral (e.g. intravenous,
subcutaneous, intramuscular, intraperitoneal, intracerebral,
intrapulmonar, intranasal or transdermal or vaginal) or enteral
(e.g., oral, or rectal) delivery according to conventional methods.
The most preferential routes of administration are intravenous and
subcutaneous administration. The formulations can be administered
continuously by infusion or by bolus injection. Some formulations
encompass slow release systems.
[0058] The coagulation factors of the present invention are
administered to patients in a therapeutically effective dose,
meaning a dose that is sufficient to produce the desired effects,
preventing or lessening the severity or spread of the condition or
indication being treated without reaching a dose which produces
intolerable adverse side effects. The exact dose depends on many
factors as e.g. the indication, formulation, mode of administration
and has to be determined in preclinical and clinical trials for
each respective indication. [0059] The coagulation factors of the
present invention can be used to treat bleedings, inducing: [0060]
All types of trauma, (blunt or penetrating, leading to severe
hemorrhage either from a single organ, a bone fraction or from
polytrauma. [0061] Bleeding during surgical procedures including
peri- or postoperative hemorrhage. [0062] Bleeding due to cardiac
surgery including patients undergoing extracorporal circulation and
hemodilution in pediatric cardiac surgery [0063] Intracerebral
hemorrhage, subarachnoid hemorrhage, sub-or epidural bleeding
[0064] Bleedings due to blood loss and hemodilution, by
non-plasmatic volume substitution leading to reduced levels of
coagulation factors in affected patients [0065] Bleedings due to
disseminated intravascular coagulation (DIC) and a consumption
coagulopathy [0066] Thrombocyte dysfunctions, depletion and
coagulopathies [0067] Bleeding due to liver cirrhosis, liver
dysfunction and fulminant liver failure. [0068] Liver biopsy in
patients with liver disease [0069] Bleeding after liver and other
organ transplantations [0070] Bleeding from gastric varices and
peptic ulcer bleeding [0071] Gynecological bleedings as
dysfunctional uterine bleeding (DUB), premature detachment of the
placenta [0072] Periventricular hemorrhage in low birth weight
children [0073] Post partum haemorrhage [0074] Fatal distress of
newborns [0075] Bleeding associated with bums [0076] Bleeding
associated with amyloidosis [0077] Hemotopoiestic stem cell
transplantation associated with platelet disorder [0078] Bleedings
associated with malignancies [0079] Infections with hemorrhaging
viruses [0080] Bleeding associated with pancreatitis
FIGURES
[0081] FIG. 1: Blood Loss (An animal model of a dilutional
coagulopathy in hypothermic, normotensive pigs by fractionated
blood withdrawal and substitution with hydroxyethyl starch (HES)
was used to investigate efficacy of the proposed treatment. A
trauma was induced by an incision into the spleen, blood loss was
determined. Substitution therapy was performed with Beriplex
P/N.RTM. and either Helixate.RTM. or Humate P.RTM.. Beriplex
P/N.RTM. is a prothrombin complex concentrate (PCC) comprising the
vitamin K dependent coagulation factors II, VII, IX and X
(Schulman, 2007). In addition Beriplex P/N.RTM. contains protein C
and protein S. Helixate.RTM. consists of recombinant FVIII as the
active constituent. Humate P.RTM., is a product consisting
essentially of FVIII as well as of vWF.)
EXAMPLE 1
Pig Model of Dilutional Coagulopathy and Spleen Trauma
[0082] Pigs were fasted overnight but had free access to water.
Animals were premedicated intramuscularly with a mixture of 2 mg/kg
Azaperone (Stresnil/Janssen), 15 mg/kg Ketamin (Ketavet/Pfitzer)
and 0.02 mg/kg Atropinsulfate (Atropinsulfate, B. Braun).
Anesthesia was induced by 10 mg/kg Thiopental-sodium via an ear
vein. Pigs were intubated and respirated via a Heyer Access
ventilator. Inhalation anaesthesia was maintained by Isoflurane
(Forene, Abbott), the concentration was 1-2%, dependent on the
status of anaesthesia.
[0083] A 1.4.times.2.1 mm catheter was advanced into the A. carotis
for the collection of blood samples and a 0.5.times.0.9 mm catheter
was placed into the A. femoralis for the continous blood pressure
measurements. A 1.4.times.2.1 mm catheter was introduced into the
V. jugularis externa for blood withdrawal and administration of
erythrocytes, plasma expander and test substances. Basic fluid
requirement was achieved by intravenous administration of Ringer
Lactate (4 mUkg x h). Body temperature was measured by a rectal
thermometer.
[0084] After a period of 30 min. to allow the stabilization of the
circulation baseline haemodynamic, coagulation and hematological
parameters were assessed (t=0). Subsequently a hypothermic,
normotensive dilution coagulopathy was induced by stepwise
withdrawal of blood. For the reinfusion of erythrocytes blood was
centrifuged (800.times.g, 10 minutes), red cells were resuspended
in NaCl to achieve the initial volume. After resuspension
erythrocytes were centrifuged again, resuspended in the half of the
initial volume in Ringer-Lactate and reinfused to the animal. After
withdrawal of about 65-70% of arterial blood, 6% Hydroxyethyl
starch (HES, Infukoll 6%, Schwarz Pharma) at room temperature were
infused intravenously.
[0085] After HES infusion blood samples were taken again (t=80
min.) and the animal was allowed to stabilize for 40 minutes. The
third blood sample was withdrawn (t=120 min.) and a standardized
spleen incision (8 cm length, 1 cm deep) was performed by a scalpel
blade.
[0086] Blood was suctioned out of the abdomen and total blood loss
and time to hemostasis was determined. Test substances were infused
shortly before the spleen incision.
[0087] Pigs undergoing the dilution procedure had a mild
hypothermia, the temperature fell from 38.5.degree. C. to
36.degree. C. Blood pressure decreased after blood withdrawal, but
was back to baseline values after the substitution with HES and
remained on a constant level until the spleen injury was performed.
Prior to spleen injury the hematocrit had decreased to 60% of its
baseline value.
Substitution Therapy
[0088] The following compounds were used for substitution therapy.
Beriplex.RTM. P/N (CSL Behring, Marburg, Germany) is a virus
inactivated human prothrombin complex concentrate (PCC). It
contains the human plasma coagulation factors II, VII, IX and X as
well as protein C and S. Humate.RTM. P (CSL Behring) is a complex
from human plasma coagulation factor VIII and von Willebrandt
factor (vWF). Haemocomplettan.RTM. (CSL Behring, Marburg, Germany)
is a fibrinogen concentrate from human plasma. NovoSeven.RTM.
(recombinant factor Vila) was from NovoNordisk. Helixate.RTM. (CSL
Behring) is a recombinant FVIII. FIXP Behring (CSL Behring) is a
FIX and FX containing product from human plasma. VWF was purified
from human plasma.
[0089] Treatment groups were as follows: 1: normal pigs (negative
control, n=5), 2: dilution control (no treatment) (n=16), 3:
Beriplex P/N.RTM. 35 U/kg (Factor IX units), 4: Beriplex P/N.RTM.
35 U/kg+Haemcomplettan.RTM. 250 mg/kg, 5: Beriplex PIN.RTM. 20-35
U/kg+Humate.RTM. 40 U/kg (Factor VIII units) (n=14) 6: Humate.RTM.
40 U/kg (n=5). 7. NovoSeven.RTM. 180 .mu.g/kg (n=5). 8.
Beriplex.RTM. P/N 30 U/kg+Helixate.RTM. (rec. FVIII) 40 U/kg (n=3),
9. FIX P+30 U/kg (Factor IX units) (n=3), 10. Beriplex.RTM.
P/N+human plasma vWF 90 U/kg (vWF units), (n=3)
TABLE-US-00005 TABLE 4 Synergistic effect of a combination therapy
using PCC and vWF Results from a Spleen Incision Trauma in Pigs:
Time to Hemostasis and Blood loss Time to Treatment hemostasis
(min.) Blood loss (mL) 1. Negative control 20.6 .+-. 7.8 83.2 .+-.
63.5 (n = 5) 2. Dilution control (n = 16) 86.7 .+-. 24.1 657.5 .+-.
298.5 3. Beriplex .RTM. P/N 37.6 .+-. 13.6 405.8 .+-. 223.1 (n = 5)
2-3 p < 0.001 4. Beriplex .RTM. P/N + 39.6 .+-. 11.9 334.5 .+-.
137.7 Haemocomplettan .RTM.P (n = 5) 2-4 p < 0.001 2-4 p <
0.05 5. Beriplex .RTM. P/N + 29.6 .+-. 7.1 190.4 .+-. 85.0 Haemate
.RTM. P (n = 14) 2-5 p < 0.0001 2-5 p < 0.00011 6. Haemate
.RTM. P (n = 5) 68.8 .+-. 27.8 471.0 .+-. 234.6 7. NovoSeven (n =
5) 91.8 .+-. 26.2 729 .+-. 265 8. Beriplex P/N + rec. FVIII 33.7
.+-. 4.6 178 .+-. 58.8 (Helixate) (n = 3) 2-8 p < 0.01 2-8 p
< 0.02 9. FIX + FX (n = 3) 115.0 .+-. 7.1 953 .+-. 232 10.
Beriplex P/N + human 27.7 .+-. 3.1 257.3 .+-. 42.6 plasma vWF 2-10
p < 0.001 2-10 p < 0.05
* * * * *